Abstract
Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.
Highlights
The yeast two-hybrid system provides an efficient method for identifying novel protein·protein interactions in small-scale studies and high-throughput screens [1,2,3]
Media made with yeast nitrogen base (YNB) from Sigma yielded the strongest signal in yeast one-hybrid experiments, whereas media made with Difco YNB yielded intermediate results, and media made with Clontech YNB produced the weakest signal (Table 1 Conditions 1-3)
We examined the impact of the media source in yeast two-hybrid experiments using a set of control plasmids, in which the T-Antigen-B42 activator chimera should bind a LexA-p53 chimera but not a LexA-Lamin chimera
Summary
Media made with YNB from Sigma yielded the strongest signal in yeast one-hybrid experiments, whereas media made with Difco YNB yielded intermediate results, and media made with Clontech YNB produced the weakest signal (Table 1 Conditions 1-3). At only 48 hours post-transformation, yeast colonies transformed with pLexA-Gal4 appeared light blue on media made with Sigma and Difco, whereas colonies on the Clontech media remained white.
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